Toxicology 313 (2013) 73–82

Contents lists available at ScienceDirect

Toxicology
journal homepage: www.elsevier.com/locate/toxicol

Tools and perspectives for assessing chemical mixtures and multiple stressors
Hans Løkke a,∗ , Ad M.J. Ragas b,c,1,2 , Martin Holmstrup a,d,3
a
Aarhus University, Department of Bioscience, Vejlsøvej 25, P.O. Box 314, DK-8600 Silkeborg, Denmark
b
Department of Environmental Science, Institute for Water and Wetland Research, Radboud University Nijmegen, P.O. Box 9010, 6500 GL Nijmegen, The Netherlands
c
School of Science, Open Universiteit, P.O. Box 2960, 6401 DL Heerlen, The Netherlands
d
Arctic Research Centre, C.F. Møllers Allé 8, bldg. 1110, DK-8000 Aarhus C, Denmark

a r t i c l e i n f o a b s t r a c t

Article history: The present paper summarizes the most important insights and findings of the EU NoMiracle project with
Received 12 March 2012 a focus on (1) risk assessment of chemical mixtures, (2) combinations of chemical and natural stressors,
Received in revised form 29 October 2012 and (3) the receptor-oriented approach in cumulative risk assessment. The project aimed at integration of
Accepted 24 November 2012
methods for human and ecological risk assessment. A mechanistically based model, considering uptake
Available online 10 December 2012
and toxicity as a processes in time, has demonstrated considerable potential for predicting mixture effects
in ecotoxicology, but requires the measurement of toxicity endpoints at different moments in time.
Keywords:
Within a novel framework for risk assessment of chemical mixtures, the importance of environmental
Chemical mixtures
Natural stressors
factors on toxicokinetic processes is highlighted. A new paradigm for applying personal characteristics
Cumulative risk assessment that determine individual exposure and sensitivity in human risk assessment is suggested. The results
Dynamic energy budget are discussed in the light of recent developments in risk assessment of mixtures and multiple stressors.
Toxicity © 2012 Elsevier Ireland Ltd. All rights reserved.
NoMiracle

1. Introduction
1.1. Background
Chemical risk assessment has traditionally focused on single
stressors. This can partly be explained by the fact that the acute
pollution problems of the 20th century were caused by a limited
number of chemicals that were emitted in relatively large quan-
tities by a limited number of well-defined sources. Consequently,
risk assessments focused on particular substances or sources. The
main questions addressed were “Can this substance cause adverse
effects?” or “Can this production plant cause adverse effects?”
These assessments typically try to capture the chain of events that
starts with the emission of a substance into the environment and
ends with the emergence of adverse health effects. This approach
has been very effective in reducing environmental problems that
are caused by single stressors and/or sources, e.g. lead, asbestos and
chlorinated insecticides.
∗ Corresponding author. Present address: Harbovad 11, DK-8600 Silkeborg,
Denmark. Tel.: +45 40 31 85 36.
E-mail addresses: hans.lokke@c.dk (H. Løkke), A.Ragas@science.ru.nl,
Ad.Ragas@ou.nl (A.M.J. Ragas), mho@dmu.dk (M. Holmstrup).
1
Tel.: +31 24 3653284.
2
Tel.: +31 45 5762679.
3
Tel.: +45 30 18 31 52.
Now the overt pollution problems have largely been solved,
awareness is growing that exposure to single chemicals is the
exception rather than the rule. In everyday life, humans and other
living organisms are rarely exposed to single stressors, but to a
mixture of different stressors; either concurrently, sequentially, or
both. Although researchers and risk assessors have always recog-
nized the need to address this problem, progress has been slow
because of lacking knowledge, experimental limitations, and scarce
funding (Callahan and Sexton, 2007).
One of the reasons for the growing interest in mixtures and
cumulative risks is that some health problems are on the rise
without an obvious explanation. It is estimated that 7.4% of the
married women in the USA between 15 and 44 years of age are
infertile (Chandra et al., 2005). The diagnoses of autism, attention
deficit and hyperactivity disorders (ADD and ADHD) and the preva-
lence of childhood brain cancer and acute lymphocytic leukemia
have all increased over the past 30 years (Jahnke et al., 2005;
Trasande and Landrigan, 2004). Air pollution and early life expo-
sure to environmental pollutants are commonly cited as possible
causes. Furthermore, a range of health issues is suspected to be
related to cumulative stress. For example, neuro-developmental
disorders can be caused by heavy metals, dioxins, PCBs and pesti-
cides (Brent, 2004); childhood cancer could be related to a number
of physical, chemical and biological agents (e.g. parental tobacco
smoke, parental occupational exposure to solvents; Trasande and
Landrigan, 2004). As a consequence, several national and inter-
national institutions have recognized the need to evaluate risks
from mixtures and multiple stressors (NRC, 1994; PCCRARM, 1997;

0300-483X/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tox.2012.11.009
74 H. Løkke et al. / Toxicology 313 (2013) 73–82
Mileson et al., 1999; US EPA, 2000, 2003; ATSDR, 2004; WHO, 2009;
European Scientific Committees, 2011).
Cumulative risk assessment can be defined as “an analysis, char-
acterization, and possible quantification of the combined risks to
health or the environment from multiple agents or stressors” (US
EPA, 2003). The conventional methods for risk assessment of sin-
gle stressors need to be adapted and extended to deal with the
specific challenges posed by mixtures and multiple stressors. This
requires new knowledge, e.g. about the complex exposure patterns
of individuals and populations, and about the potential interactions
between stressors. Novel exposure tools are required that focus
on the exposed individual or population, instead of the traditional
tools that focus on single substances or pollution sources (Loos
et al., 2010a). Measurement data on exposures to mixtures and
multiple stressors need to be combined with data on the preva-
lence of adverse health effects in populations and ecosystems in
order to identify potential causal relationships (Rappaport, 2011).
Furthermore, methods are needed to predict the effects of concur-
rent or sequential exposures to mixtures and multiple stressors,
preferably based on data from single compounds because these are
widely available. We need to understand how and why stressors
interact because it is not feasible to test all possible combinations
of stressors in the laboratory. Those tests that are necessary need
to be performed according to intelligent testing strategies to opti-
mize the use of resources and limit the use of laboratory animals.
Collaboration between human and ecological experts in toxicology
and risk assessment is desirable because both fields struggle with
similar issues and can benefit from each other’s strengths (Ragas
et al., 2011a). In addition to interactions between chemicals, there
are possible interactions with other stressors, such as climate vari-
ables (Schlink et al., 2002; Leitte et al., 2009), which need to be
addressed.
1.2. The EU NoMiracle project
In November 2004, the EU research project NoMiracle was
launched (http://www.nomiracle.jrc.ec.europa.eu). The main aim
of the project was to gather new knowledge for the improve-
ment of current risk assessment methods and the development
of novel methods for cumulative risk assessment. The work was
conducted until 30 October 2009. Thirty eight institutions from
17 European countries participated in the project. The consortium
consisted of scientists with expertise in human toxicology, epi-
demiology, aquatic and terrestrial ecotoxicology, risk assessment,
environmental chemistry, biochemistry, toxicogenomics, physics,
mathematical modeling, geographical informatics, and social and
economic sciences. Box 1 shows the main goals of the NoMiracle
project. The themes of NoMiracle were rather broad, allowing for
interdisciplinary collaboration.
The project resulted in more than 200 scientific reports and
200 peer-reviewed papers covering a large diversity of topics
such as the identification of exposure scenarios, sorption behav-
ior of substances, environmental degradation, analytical methods,
fate modeling, toxicokinetics, mixture toxicity, effects of multi-
ple stressors, in vitro test systems, probabilistic risk assessment
methods, exposure modeling, risk perception and risk visualiza-
tion. Six open international workshops and conferences were held.
An international workshop on mixture toxicity was organized
together with the Society of Environmental Toxicology and Chem-
istry (SETAC) and resulted in a book on mixture toxicity (Van
Gestel et al., 2010). The main results of the NoMiracle project
were published as a special issue of Science of the Total Envi-
ronment (Løkke et al., 2010). The new methods and models that
were developed are publicly available in the NoMiracle Tool Box
(http://www.nomiracle.jrc.ec.europa.eu/Lists/Toolbox/).
Box 1
Goals of the EU NoMiracle project.
1. To develop new methods for assessing the cumulative risks
from combined exposures to several stressors including
mixtures of chemical and physical/biological agents.
2. To achieve more effective integration of the risk analysis of
environmental and human health effects.
3. To improve our understanding of complex exposure sit-
uations and develop adequate tools for sound exposure
assessment.
4. To develop a research framework for the description and
interpretation of cumulative exposure and effect.
5. To quantify, characterise and reduce uncertainty in current
risk assessment methodologies, e.g. by improvement of the
scientific basis for setting safety factors.
6. To develop assessment methods which take into account
geographical, ecological, social and cultural differences in
risk concepts and risk perceptions across Europe.
7. To improve the provisions for the application of the precau-
tionary principle and to promote its operational integration
with evidence-based assessment methodologies.
The present paper summarizes the most important insights and
findings of the EU NoMiracle project within three major fields
of the project, namely: (1) risk assessment of chemical mixtures,
(2) combinations of chemical and natural stressors, and (3) the
receptor-oriented approach in cumulative risk assessment. The
results of the NoMiracle project are discussed in the light of recent
developments in risk assessment of mixtures and multiple stress-
ors. The paper concludes with an outlook for the future and a
number of research and policy recommendations.
2. Risk assessment of chemical mixtures
2.1. Mixture experiments
The NoMiracle consortium set up a series of (eco)toxicity tests
with chemical mixtures. The chemicals were taken from a prior-
ity list that focused on metals, polyaromatic hydrocarbons (PAHs)
and specifically acting pesticides (e.g. organophosphate and neoni-
cotinamide pesticides). Species used for testing included the Vibrio
fischeri MicrotoxTM system, Tubifex tubifex, chironomidae, Danio
rerio, Daphnia magna, Mytilus galloprovincialis, Lumbricus rubel-
lus, Folsomia candida, Caenorhabditis elegans, carabid beetles and
human cell lines (human A549 cells and Jurkat cells) including
existing and custom generated reporter cell lines. Measured end-
points included cellular change, weight change, cell proliferation,
reproduction, growth and viability (Oostingh et al., 2008, 2009;
Röder-Stolinski et al., 2008a,b).
The underlying idea was to explore the application range of two
predictive models for the joint effects of two or more substances,
i.e. concentration addition (CA) and independent action (IA), and
deviations from these descriptive models, including greater than
additive or less than additive toxicity, dose level or dose ratio
dependent deviations. The broad applicability of these two ref-
erence models has been demonstrated for both similarly acting
(Altenburger et al., 2000; Deneer et al., 1988a; Faust et al., 2001;
Hermens et al., 1985; Howard et al., 2010) and independently act-
ing compounds (Backhaus et al., 2000; Deneer et al., 1988b; Faust
et al., 2003). Data on the performance of these models can serve
multiple purposes. First, it can be used to assess the probability
that the two reference models fail to correctly describe the joint
effects of chemicals, which is relevant information for risk asses-
sors, e.g. when deriving a safety factor for mixture toxicity. Second,
 H. Løkke et al. / Toxicology 313 (2013) 73–82
exploration of those cases that deviate may reveal patterns leading
to a better mechanistic understanding of mixture effects. For exam-
ple, it could be investigated whether deviations are conserved over
different taxonomic groups or endpoints, and which contaminant
groups and stressors show the greatest propensity for deviations.
Ultimately, NoMiracle generated a database of a 322 experimen-
tal data-sets, i.e. 144 studies with ecotoxicological test species and
178 with human cell lines (e.g. Gomezeyles et al., 2009; Loureiro
et al., 2010; Martin et al., 2009). A preliminary analysis of the eco-
toxicological data with an updated version of the MixTox model
(Jonker et al., 2005) indicated that 94 mixtures (65.3%) had signifi-
cant interaction, i.e. greater than additive (16.0%), less than additive
(17.3%), dose-level dependent deviation (16.0%) and dose ratio
dependent deviation (16.0%). These numbers are in good agree-
ment with previous studies. For example, Cedergreen et al. (2008)
found interactions in 50% of the cases included in a separate mixture
effect program, and Warne (2003) reported 20–30% nonadditive
cases based on a review of ecotoxicological mixture studies. The
significance of the observed deviations from the CA and IA models
is discussed in Section 5.
Creation and exploration of the NoMiracle mixture database
proved more difficult than originally anticipated. The mixture
experiments took more time and the researchers wanted to publish
their results before inclusion in a database. As a result, the database
was incomplete and only preliminary analyses could be performed.
The partners are currently looking for fundings to further extend
the database because it has huge potential, e.g. to explore the mag-
nitude of the deviations and compare the results with data from
Ross (1996) and Ross and Warne (1997) who reported that 5%
of the mixtures included in a review differed more than a fac-
tor of 2.5 from CA, and 1% differed more than a factor of 5. This
type of data could be used to derive a safety factor for mixture
toxicity. Other potential applications of the database include: (1)
verification whether interactions at low dose levels are occur-
ring or toxicologically insignificant, or not (European Scientific
Committees, 2011); (2) verification whether the range of devia-
tion from additivity decreases as the number of components in a
mixture increases (Warne and Hawker, 1995); (3) explore whether
data about metabolic interactions can be used to predict mixture
toxicity (Ragas et al., 2011b).
2.2. Mechanistic mixture studies
Mixture experiments can provide an indication of the joint
effects of substances, but it is practically infeasible to test all mix-
ture combinations. The number of possible substance combinations
is sheer endless and the dose–response relationship of a certain
combination is likely to depend on the dose ratios. Therefore, a
mechanistic approach to assessing mixture risk is highly desirable.
If the most important mechanisms involved in mixture toxicity can
be identified and described, this knowledge can be used to improve
the prediction of mixture risks.
To provide a basis for interpreting the mechanisms involved in
mixture toxicity, a biologically based framework was developed
(Fig. 1; Spurgeon et al., 2010). The framework consists of three
phases: the external exposure phase, the toxicokinetic phase and
the toxicodynamic phase. The first phase involves interactions in
environmental media that may influence the external dose of one
or more chemicals, and includes processes such as speciation, bind-
ing and transport (Spurgeon et al., 2011). The second phase involves
interactions inside the organism that influence the internal dose of
one or more chemicals and includes processes such as absorption,
metabolism and elimination (Svendsen et al., 2011). The last phase
involves interactions at the target site that may modulate the toxic
impact of one or more substances (Svendsen et al., 2011). Once
the nature and type of potential interaction has been established,
75
designing experimental approaches that can investigate the under-
lying mechanisms is made easier.
The framework in Fig. 1 built as a background for the mech-
anistic mixture studies in NoMiracle (e.g. Dimitrov et al., 2005;
Dimitrova et al., 2010; Dorne, 2007; Vandenbrouck et al., 2009).
Part of the work focused on the development of a process-based
approach for describing and predicting mixture effects (Baas et al.,
2010a) based on the theory of dynamic energy budgets (DEBs)
(Kooijman, 2009). The approach considers toxicity as a process over
time that depends on (1) the buildup of the internal concentration
which is modeled using a one-compartment model, and (2) a haz-
ard model that describes the adverse effect as a chance process, e.g.
using a killing rate. The mixture effect is build up from the effects
of the individual components and all possible binary interactions
in the mixture. The approach results in a mathematical model that
consists of three parameters per compound (the no effect concen-
tration, an elimination rate and an effect rate), a control effect rate
for the experiment (to account for the effect rate in the unexposed
control group) and one interaction parameter for each pair of com-
pounds. The parameter values can be determined by fitting the
model to a time series of toxicity data, e.g. data on survival. The
model has shown good performance in several case studies, e.g.
modeling the effect of binary metal mixtures on survival of the soil
arthropod F. candida (Baas et al., 2007) and modeling the effect of a
mixture of 14 PAHs on the survival of the fish Pimephales promelas
(Baas et al., 2009a). The model was also used to predict the effect
of surface waters contaminated with over 90 chemicals on the sur-
vival of D. magna (Baas et al., 2009b). In 34 out of 37 cases (92%),
daphnid survival was predicted correctly.
The approach can be considered a step forward compared to
the conventional CA and IA models because it has a mechanis-
tic basis and considers uptake and toxicity as processes in time
(Baas et al., 2010b). Consequently, the model uses relatively few
parameters which actually have a biological meaning (e.g. killing
and elimination rates). Furthermore, the model is less sensitive
to random variations because the entire data-set is fitted to the
model, whereas the CA and IA models have to be fitted separately
for each moment in time. The approach has considerable poten-
tial for describing and predicting mixture effects, but requires the
measurement of toxicity endpoints at different moments in time.
The extra costs of performing toxicity measurements in time come
with the promise of an increased understanding of the underlying
biological mechanisms and prediction of mixture effects.
3. Combinations of chemical and natural stressors
In real-world environments it is not unusual for organisms and
humans to be exposed to several stressful factors of natural origin
besides those of polluting chemicals. These natural factors may in
aquatic ecosystems include anoxia and pH changes which have seri-
ous consequences for many species (Hoegh-Guldberg et al., 2007).
In the terrestrial environment extreme climatic conditions, such as
heat waves, drought, flooding or extreme cold are also important
stressors. With current concerns over global climate changes pre-
dicted to increase the frequency and amplitude of extreme weather
events (IPCC, 2007) it is important to include interactions between
multiple stressors in modern risk assessment procedures. Effects
of chemical pollution may interact with the effects of these natu-
ral abiotic stressors (Laskowski et al., 2010; Heugens et al., 2001;
Gordon, 2001). In addition, biotic stressors such as parasites and
pathogens may interact with environmental toxicants (Holmstrup
et al., 2010). This also applies to humans since environmental toxi-
cants may compromise the human immune system (Oostingh et al.,
2008). In the NoMiracle project, it has been found that several of
the chemicals tested had a clear immunomodulatory effect at non
76 H. Løkke et al. / Toxicology 313 (2013) 73–82
Fig. 1. Framework for the mechanistic interpretation of mixture effects (adapted from Spurgeon et al., 2010). C1 and C2 indicates two different compounds interacting with
soil particles, biological macromolecules or structures, and ligands (L) in a mixture situation.
cytotoxic concentrations (Holmstrup et al., 2010). This suggests
that an activated immune system can be more prone to react with
environmental toxicants and that the concentrations of pollutants
that might have an impact on human health are lower when the
person exposed is ill.
The approach used in studies of multiple stressors (natural and
chemical) is based on the same conceptual framework as out-
lined for mixture effects in Fig. 1. Natural factors can influence
the bioavailability of chemicals in many ways (left side of Fig. 1).
Examples include water chemistry, which can alter the bioavail-
ability of heavy metals in aquatic ecosystems (Weber et al., 1992;
Santore et al., 2002), and air humidity influencing the occurrence of
airborne particles, which in turn may have an effect on the preva-
lence of chronic bronchitis among people in areas with heavy air
pollution (Leitte et al., 2009). The list of examples in the litera-
ture is extensive (see for example review by Heugens et al., 2001),
but at present these issues are only included to a limited extent
in risk assessment procedures despite their apparent importance.
Toxicokinetics may also be drastically influenced by environmental
factors. A prime example is temperature, which may both influ-
ence the active uptake and elimination rate of toxicants, but this
primarily applies to poikilothermic organisms where metabolism
is directly coupled to temperature (but see also review by Gordon,
2001). On the one hand, poikilothermic organisms such as insects
are often more active at higher temperatures and may thus take
up more of a toxicant (e.g. insecticides) than at low temperature
where activity is depressed. On the other hand, metabolism and
detoxification processes may run faster at the higher temperatures
(Talent, 2005). Andersson and Förlin (1992) reviewed studies on
biotransformation of organic hydrocarbons (e.g. PAHs) in fish and
concluded that induction and efficiency of the cytochrome P450
system is highly dependent on ambient temperature, although no
consistent pattern due to either rising or declining temperature
was found. However, these examples highlight the importance of
environmental factors on toxicokinetic processes.
Last, but not least, interactions between effects of natural stress-
ors and effects of toxicants can occur at the target receptor, or
more broadly spoken, at the physiological level (right-hand side
of Fig. 1). Results of the NoMiracle project have shown that greater
than additive effects are not unusual when organisms are exposed
to combinations of natural stressors and toxic stressors, especially
when the natural stressor reaches extreme levels; a review of more
than 140 studies showed that about 50% of studies on invertebrates
revealed some degree of greater than additive interactions taking
place (Holmstrup et al., 2010; Laskowski et al., 2010). For example,
sublethal concentrations of copper, which is a widespread terres-
trial contaminant, can completely destroy the ability of earthworms
to withstand frost or drought (Bindesbøl et al., 2005; Fisker et al.,
2011). Other contaminants may significantly reduce the drought
tolerance of soil organisms such as Collembola (Højer et al., 2001;
Holmstrup, 1997; Skovlund et al., 2006). Screenings of several
environmental contaminants suggest that pollutants where the
mode of action is connected with membrane stability and func-
tioning (e.g. copper, non-ionic detergents, and several PAHs) can
reduce the drought and cold tolerance of poikilothermic animals
(Holmstrup et al., 2010). As a corollary to this it can be argued that
cold and drought can increase the negative effects of environmen-
tal pollutants. The main reason for this interaction may be that
such pollutants cause changes in the composition of membrane
phosholipids via oxidative stress and lipid peroxidative processes,
altering the physical properties of the membranes (Bindesbøl et al.,
2009), or merely change the physical properties of cellular mem-
branes by their presence in, and interactions with the lipid matrix
(Ogunbayo et al., 2007; Van Wezel et al., 1996; Jørgensen et al.,
1991). Both phenomena may potentially compromise membrane
functionality with respect to challenging osmotic or thermal con-
ditions. However, published research in this area is still mainly of a
phenomenological and descriptive character; documented mecha-
nistic and molecular understanding remains to be provided.
It is obvious that organisms in the environment and humans
are well adapted to harsh environmental conditions, and thus sig-
nificant interactions with toxicants are likely a rather stochastic
phenomenon that does not occur on a regular basis. However, it
seems reasonable to conclude that situations can arise that are
potentially causing so large effects that risk assessment should
somehow account for these situations. As an example, the Euro-
pean heat wave in 2003 caused significant increase in mortality
especially in the French population. It is not known how much of
this extra mortality was related to simultaneous pollution prob-
lems, but it illustrates that under severe stress humans and other
 H. Løkke et al. / Toxicology 313 (2013) 73–82 77

resulting from human activities. In a similar way the European

Without pollution
Framework Directive by monitoring a range of biotic communi-
ties potentially addresses complex mixtures of stressors in very
Response different regions and water body types (Hering et al., 2010).

4. The receptor-oriented approach

Critical stress
Risk management aims to control events that can result in
adverse effects in humans or ecosystems. In the case of single

level
chemicals, these events can be presented by a single chain, start-
ing with the production and emission of the chemical of concern
Adding pollution and ending with the adverse effects (Fig. 3). When the relationships
between the links of the chain are known, data and measures relat-
ing to one link can be translated relatively easily into implications
Response

for other links. This explains why traditional risk assessment often
focuses on substances or sources.
In the case of multiple stressors, the events that lead to adverse
Critical stress

effects can no longer be presented by a single chain, but multiple

chains are necessary that converge at the receptor, i.e. the individ-
ual, population or (eco)system that is being exposed (Fig. 3). The
level

Increasing natural stressor
Fig. 2. Performance of an organism is related to natural stress factors (“stressors”) in
a normal dose–response relationship. For all stressors, organisms are able to com-
pensate for deleterious effects until a certain “critical stress level” is reached; at
higher levels performance declines (upper panel). If additional stressors such as
pollution is added to the system, the critical stress level may be shifted to the left
(lower panel) indicating that pollution has made the organism less tolerant toward
the natural stressor.
organisms may become more susceptible to other types of stress
than they usually are (Fig. 2).
Compared with human risk assessment, it is more common
in ecological risk assessment to include natural stressors. Thus,
the European Marine Strategy Framework Directive (MSFD, 2008)
takes into account the structure, function and processes of the con-
stituent marine ecosystems together with natural physiographic,
geographic, biological, geological and climatic factors, as well
as physical, acoustic and chemical conditions, including those
branched nature of this chain implies that data and measures relat-
ing to one link can no longer be easily translated into implications
for other links. There is a strong impetus to focus on the receptor
as an integrator of exposure and effects over space and time. The
question is no longer “How does this substance or source influ-
ence public health or ecosystems?”, but “How are public health
and ecosystems influenced by the mixture of stressors they are
being exposed to?”. Within the NoMiracle project it was concluded
that cumulative risk assessment requires a paradigm shift from
the traditional stressor- and source-oriented assessments toward
receptor-oriented assessments. This paradigm shift has some seri-
ous implications for the way exposure and effects are assessed
and requires a broader regulatory context than currently provided.
These issues are discussed in more detail below.
4.1. Exposure assessment
An important challenge in receptor-oriented exposure mod-
eling is the description of the activity pattern of the receptors,
including movement. Two approaches can be distinguished, i.e. a

Stressor 1 Receptor
Environmental
Source(s) Emission(s) Exposure Effects
Quality

Stressor 1
Environmental
Source(s) Emission(s)
Quality

Stressor 2
Environmental
Source(s) Emission(s)
Quality Receptor
Exposure Effects

Stressor X
Environmental
Source(s) Emission(s)
Quality

Fig. 3. The chain of events that can result in adverse effects of stressors in humans or ecosystems. The process is illustrated for single stressors in the upper panel and for
multiple stressors in the lower panel.
78 H. Løkke et al. / Toxicology 313 (2013) 73–82
descriptive and a predictive approach. In the descriptive approach,
data about activity patterns are gathered, stored in a database and
subsequently used for modeling exposure. This approach is typical
for human exposure models such as LifelineTM (The Lifeline Group
Inc., 2007), CARES (ILSI, 2008), SHEDS (Zartarian et al., 2006, 2007)
and TRIM.Expo/APEX (US EPA, 2006a,b). Important strengths of the
descriptive approach are the simulation of realistic activity and
movement patterns and the preservation of correlations between
different activities. However, the exposure predictions only apply
to the conditions reflected in the database. The approach does not
account for changes that may influence the activity patterns such as
the introduction of a new transport system or a new food product
on the market.
In the predictive approach, activity patterns are modeled using
behavioral rules such as the preference of a species for a cer-
tain habitat or for a certain type of food. This approach is typical
for wildlife exposure models such as SE4 M (Hope, 2005), ALMaSS
(Topping et al., 2003) and EcoSpace (Loos et al., 2010b). These
models use object-oriented programming techniques to simulate
interactions between organisms and between organisms and the
environment. An important strength is the large explanatory power
of these models, i.e. the activity pattern is an emergent property
that depends on the characteristics of the receptor, environment
and behavioral rules. However, the accuracy of the predictions
strongly depends on the quality of the behavioral rules which rarely
have been validated.
Loos et al. (2010a) compared five human and five wildlife
receptor-oriented exposure models. They concluded that human
models can act as a source of inspiration for wildlife models when
it comes to the compilation and use of data on activity patterns. A
nice example is a recent Dutch study on the foraging behavior of
the eagle owl (Bubo bubo), which unexpectedly revealed that these
birds spend an important part of their time foraging in the city
of Maastricht (Houben, 2011). This results in an entirely different
exposure pattern than would be predicted with random behavioral
algorithms. Loos et al. (2010a) furthermore suggested that wildlife
models can serve as a source of inspiration for human models when
it comes to the simulation of emergent activity patterns. This sug-
gestion was taken up by Schlink and Ragas (2011) who compared
different algorithms to simulate human movement in urban areas.
Of the algorithms tested, truncated Lévy flights (TLFs) and agenda-
based walks performed best because they provide conditions for
individual-specific trajectories and exposure. They demonstrated
the plausibility of their results for exposure to airborne benzene
and the combined exposure to benzene and nonane.
We conclude that cumulative risk assessment requires further
development of receptor-oriented exposure models. Important
issues to address include (1) gathering of measurement data
on activity and movement patterns, (2) development of reli-
able algorithms to simulate activity patterns and movement, (3)
maintenance of correlations between different activities, and (4)
further development and application of object-oriented program-
ming techniques in exposure modeling.
4.2. Effect assessment
Challenges in receptor-oriented effect assessment include (1)
exploration of the impact of temporal variations in exposure
regimes, and (2) inclusion of receptor characteristics in effect
assessment. Receptor-oriented exposure models typically result in
a graph that reflects the variation in exposure(s) over time. This
triggers the question how to deal with temporal variations in expo-
sure(s). Most toxicity tests are performed under constant exposure
conditions. Extrapolation to time-variant exposures is relatively
simple for substances that accumulate or for which the effect
accumulates, but can be more problematic for acute toxicants.
This requires a good understanding of the toxicokinetic processes
involved. It becomes even more complicated when different stress-
ors are involved, because the order of the different exposures
may affect the outcome. It is remarkable how little systematic
knowledge is available about the impact of time-variable exposure
conditions, especially in relation to different stressors (e.g. Johnson
et al., 1990; Kimizuka et al., 1987; Kimizuka and Hayashi, 1993).
Here lies an enormous challenge for the future. The homogeneous
exposure regimes currently applied in toxicity tests will have to be
extended to include the effects of the time-variable and sequential
exposures that occur in real life. Some data on sequential exposure
are available e.g. from studies in carcinogenesis (Kortenkamp et al.,
2009). Only very few environmental studies have been conducted
using sequential exposure, e.g. Drost et al., 2003; Vallotton et al.,
2009, focussing on pulse exposure of algae to herbicides.
The sensitivity of individuals for a stressor typically varies
within a population. When deriving a safe exposure level for a
population, this inter-individual variability is traditionally
addressed by applying a safety or assessment factor (Dorne,
2010). The resulting value aims to protect sensitive individuals
within the population. This approach is suitable for single stressors,
but seems less suitable for multiple stressors. For one reason, the
approach will result in overly conservative risk assessments when
the sensitivities for different stressors are uncorrelated (Price et al.,
2009). However, when sensitivities are correlated, as is the case for
substances that are metabolized by the same metabolic pathway,
the approach is appropriate. This issue could be addressed by
including the personal characteristics that determine individ-
ual sensitivity in the effect assessment. The receptor-oriented
approach provides a perfect platform to realize this. Several large
scale studies are currently being performed in which individual
data on activity patterns, chemical exposures, personal character-
istics (e.g. ethnicity, socio-economic status and genetic profile) and
health status are gathered in a coherent way, e.g. the National Chil-
dren’s Study in the USA (http://www.nationalchildrensstudy.gov/).
These studies will provide a wealth of information that will enable
the integrated modeling of exposure and effects in a truly receptor-
oriented approach. With this type of approach it should be possible
to identify more realistic combinations of activity patterns and
personal characteristics that result in high risks for single as well
as multiple stressors.
4.3. Regulatory context
In the human health area, mixture issues are often addressed
based on legislation and regulations that either focus on (indi-
vidual) substances or compartments. For example, widely used
common mixtures such as PAHs and PCBs are evaluated within the
context of substance-specific regulations such as the Toxic Sub-
stances Control Act (TSCA) in the USA and the REACH program
in the EU. Mixtures encountered at polluted sites are evaluated
within the context of regulations such as Comprehensive Environ-
mental Response, Compensation, and Liability Act (CERCLA) in the
USA dealing with improperly deposited wastes, and the Water and
Soil Framework Directives in the EU. These regulations do allow
for assessment of mixture risks, but the problem definition is gen-
erally limited to a single compartment or a common mixture, e.g.
exposure to contaminated soil or to PAHs. However, the nature of
mixture and cumulative risks can be much broader, e.g. a farmer
who works with pesticides and lives on a metal-polluted soil in
a house with high radon exposure levels. Indeed, it is interesting
to note that comprehensive regulations to protect human health
against the potential effects of sequential or concurrent exposure
to multiple stressors are currently lacking. The identification and
regulation of mixture risks requires a broader regulatory context
than currently covered by most regulations. The focus should not
 H. Løkke et al. / Toxicology 313 (2013) 73–82
be on a specific substance, mixture or compartment, but on the inte-
grated protection of human or ecological receptors from exposure
through different routes and at different moments in time.
Contrary to the human health area, legislation concerning the
protection of ecosystems and wildlife is much more integrative.
The focus is on protection of the receptor against different types
of stressors. Mandated by acts like the Endangered Species Act
(ESA) and the Marine Mammal Protection Act (MMPA), govern-
mental agencies such as the US Environmental Protection Agency
are required to assess risks to ecosystems and wildlife populations
resulting from exposure to anthropogenic stressors in the broadest
definition (e.g. agricultural and urban land use, introduced invasive
and exotic species, nutrient enrichment, direct human disturbance,
and toxic chemicals; Munns, 2006). These regulatory laws promote
risk assessment to consider both chemical and other (physical and
biological) stressors.
5. Discussion
The NoMiracle project covered a broad field. In this review we
have focused on three important topics for the assessment of risks
associated with multiple stressors, namely the need for novel tools
for (1) risk assessment of chemical mixtures, (2) combinations
of chemical and natural stressors, and (3) the receptor-oriented
approach in cumulative risk assessment.
Within the risk assessment of chemical mixtures, there is much
discussion on the occurrence and frequency of nonadditive effects,
defined as deviations from a simple additive model. Recently Boobis
et al. (2011) analyzed key studies from 1990 to 2008 relating to
greater than additive interactions in mammalian test systems at
low doses. Of 90 studies, they identified 6 studies that provided
useful quantitative estimates of greater than additive effects at low
doses, and the predicted deviations from additive models were less
than a factor of 4. Within ecotoxicology, a higher frequency of devi-
ations is reported. As a rule of thump the frequency seems to lay in
the interval of 20–65% (Cedergreen et al., 2008; Jonker et al., 2005;
Warne, 2003). The deviations were in the same order (a factor of
2–5) as found by Boobis et al. (2011).
These deviations were found using the CA and IA models with
concentrations at relatively high dose levels. In the NoMiracle
project, the use of the CA and IA models gave rise to concern when
analyzing ecotoxicological data from studies with measurements
at intermediate time points. Such data sets are rare, however. Baas
et al. (2007) reported a study on the survival of springtails (F. can-
dida) which were exposed to binary mixtures of metals. Depending
on the time of observation, deviations from the study with cop-
per and cadmium using the CA model changed between significant
greater than additive effects, dose level dependent effects greater
than additive, dose ratio dependent interaction and no interaction.
At one time point and at high doses, less than additive effects were
observed. The analysis by use of the IA model showed greater than
additive effects at all time points. By analyzing the same data with
the newly developed process-based model using the DEB theory,
the authors showed excellent agreement between measured and
calculated survival data. Only in the case of a mixture of copper and
lead was a slight less than additive effect found; the other mixtures
showed no interaction. The authors concluded that when models
do not incorporate time, it is impossible to speak of interaction in
the mixture because the interaction depends on the time point that
is considered. This conclusion has been underpinned by a series of
experiments (Baas et al., 2009a,b, 2010a,b; Jager et al., 2010).
The lesson learned from the NoMiracle project on simple addi-
tion models such as the CA and IA models is that they may be useful
for screening purposes and for analysis of groups of chemicals with
similar mode of action. However, the time factor may be crucial
79
for a proper assessment, in particular in cases of sequential expo-
sure. The models based on DEB theory have the basic assumption
that toxicants must be taken up by the organism before they can
exert an effect. The internal concentration determines the effect,
and once it is built up above a certain threshold level effects start
to show. This threshold level is defined as the NEC (the no effect
concentration). It has been shown that the NEC is shared by toxi-
cants having the same mode of action as for example PAHs causing
baseline toxicity (Baas et al., 2010c). This allows for reduction in
experimental effort in assessing effects of mixtures, extrapolation
to other mixtures, other points in time, or in a wider perspective to
other organisms.
When dealing with the impact of multiple stressors, the assess-
ment is most often restricted to chemical mixtures. The reason is
obviously that it is more complicated to deal with natural stress-
ors, and no approved methods are at hand. Given that exposure
to multiple chemicals from different sources via multiple routes is
beyond the scope of present regulations, it is far more distant to
include natural stressors. However, it is problematic to ignore the
natural stressors if one wants to unravel the causes of increased
rates of disease or adverse environmental impacts that may be
wholly or partially caused by chemical substances. In many cases,
the interactions between natural and anthropogenic influences
need to be examined. For human health, obvious examples are the
interactions of exposure to chemicals in daily life and the work
environment with natural stressors such as diseases, biologically
active components in food, UV radiation, alpha particles from radon
or exposure to gamma-radiation. In the environment, natural stress
factors are often more important and widespread than stress from
chemical pollution, which is typically rather local and related to
industrial or agricultural activities (e.g. Kozlov and Zvereva, 2007).
Natural stressors are particularly important in extremely hot, cold
or dry environments such as the polar regions, or deserts, where
organisms and humans must cope with recurring climatic stress
(Wharton, 2002), but the global changing of climate and increased
occurrence of extreme weather events such as heat waves (IPCC,
2007) will also increase the risk from combined effects of pollu-
tion and natural stress factors. Our point is that effects of natural
stressors can be amplified by interaction with the chemicals.
During the recent years, the question of how to manage the risk
from multiple chemicals has come into focus. There are no gener-
ally applicable guidelines as to when assessment of combinations
of chemicals should be carried out. In part this is due to the assump-
tions that substantial effects due to exposure to a combination of
chemicals are uncommon (European Scientific Committees, 2011).
The EU Advisory Committees concluded that for chemicals with
different modes of action (i.e. acting independently), no robust evi-
dence is available that exposure to a mixture of such substances
is of health concern if each individual chemical is present at or
below their no effect levels. They recommend an approach for
the assessment of chemical mixtures which emphasizes the need
for information on the mode of action, but the decision algorithm
comes to a stop if the Threshold of Toxicological Concern for sin-
gle substances is not exceeded. This approach may be questioned
in cases where chemicals modify the absorption of others (e.g.
skin penetration enhancing substances in cosmetics) or chemicals
competing for active transport mechanisms (uptake, clearance).
Independently acting chemicals in a mixture may also modify the
metabolism of other mixture components, e.g. if two or more sub-
stances are metabolized by the same enzyme, thereby enhancing
the effect of single compounds at the Threshold of Toxicological
Concern.
Within the WHO and IPCS a framework has been developed for
risk assessment of combined exposure to multiple chemicals using
a tiered approach (Meek et al., 2011). This framework builds on
previously published guidance for priority setting and assessment
80 H. Løkke et al. / Toxicology 313 (2013) 73–82
of combined exposures (e.g. US EPA, 2007). There is a gradual tran-
sition from the risk assessment of single compounds to multiple
compounds by multiple routes (European Scientific Committees,
2011), to multiple compounds by multiple routes taking individ-
ual variation into consideration (US EPA, 2007), and finally to
the receptor-oriented approach (see Section 4). By the receptor-
oriented approach the individual is assessed for the total effect of
multiple stressors, i.e. not only low doses of pollutants. For example,
current risk assessment is concerned with traces of pharmaceuti-
cal drugs in drinking water, while an often long-lasting exposure
to medical products is overlooked. In the use of drugs, the empha-
sis is usually on the benefits and to a lesser degree on the side
effects, and rarely on interactions with xenobiotics. In a similar way,
the interactions of xenobiotics with deposits of persistent organic
compounds in human fat, radon radiation, tobacco smoke, alco-
hol and toxic food components are not accounted for. There are
ethical, political and practical reasons not to include these major
impact factors. However, to understand the occurrence and even
increase in frequency of a series of chronic diseases and the role of
xenobiotics, the whole picture needs to be taken into account.
Future testing for mixture effects will take advantage of the
ongoing revolution in biology and biotechnology. In NoMiracle,
studies on the potential for using omics in the assessment of
chemical mixtures was initiated by use of marine animals, e.g.
by Dondero et al. (2010) and Dorne (2010). Within human biol-
ogy, new methods are being developed that use biological data
in order to find biochemical pathways that are relevant to the
different responses of an organism to different conditions. Bio-
chemical pathways, instead of being treated as just sets of genes, are
viewed as a network of interactions between proteins or metabo-
lites (Thomas et al., 2009). By this approach, the identification of
key regulatory pathways that integrate genetic and environmen-
tal modulators defines disease associated targets that will allow
for efficient screening of large numbers of environmental factors
(Gohlke et al., 2009). Such novel methods and approaches are
anticipated to play a major role in future risk analyses of multiple
stressors by multiple routes using a receptor-oriented approach.
Conflict of interest
None.
Funding source
EU FP6 integrated project NoMiracle Contract No. 003956.
Acknowledgements
The studies were supported by the EU 6th Framework program
for Research and Technological Development under the Integrated
Project NoMiracle (Novel Methods for Integrated Risk assessment
of Cumulative Stressors in Europe; http://nomiracle.jrc.it), Contract
No. 003956.
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